The lactate sensor GPR81 regulates glycolysis and tumor growth of breast cancer

Metabolic reprogramming is a malignant phenotype of cancer. Cancer cells utilize glycolysis to fuel rapid proliferation even in the presence of oxygen, and elevated glycolysis is coupled to lactate fermentation in the cancer microenvironment. Although lactate has been recognized as a metabolic waste product, it has become evident that lactate functions as not only an energy source but a signaling molecule through the lactate receptor G-protein-coupled receptor 81 (GPR81) under physiological conditions. However, the pathological role of GPR81 in cancer remains unclear. Here, we show that GPR81 regulates the malignant phenotype of breast cancer cell by reprogramming energy metabolism. We found that GPR81 is highly expressed in breast cancer cell lines but not in normal breast epithelial cells. Knockdown of GPR81 decreased breast cancer cell proliferation, and tumor growth. Mechanistically, glycolysis and lactate-dependent ATP production were impaired in GPR81-silenced breast cancer cells. RNA sequencing accompanied by Gene Ontology enrichment analysis further demonstrated a significant decrease in genes associated with cell motility and silencing of GPR81 suppressed cell migration and invasion. Notably, histological examination showed strong expression of GPR81 in clinical samples of human breast cancer. Collectively, our findings suggest that GPR81 is critical for malignancy of breast cancer and may be a potential novel therapeutic target for breast carcinoma.


GPR81 is expressed in breast cancer cells.
To determine the pathophysiological roles of GPR81 in breast cancer cells, we first compared the expression level of GPR81 in the tumorigenic human breast cancer cell lines MCF7 and MDA-MB-231 with that in the non-tumorigenic breast epithelial cell line MCF-10A 27 . Western blotting analysis demonstrated that MCF-7 and MDA-MB-231 cells had higher levels of GPR81 protein than MCF-10A cells (Fig. 1a). Immunofluorescent analysis revealed that GPR81 was expressed on the cell membrane of MCF-7 and MDA-MB-231 cells and co-expressed with the cell membrane marker Na/K ATPase (Fig. 1b). In contrast, GPR81 expression was not detectable on the cell membrane of MCF-10A cells (Fig. 1b). The expression of the lactate transporter MCT4 was increased in MCF-10A and MDA-MB-231 cells compared with that in MCF-7 cells (Fig. 1a). In contrast, the expression of MCT1 was highest in MCF-7 cells among these three cell lines (Fig. 1a). Interestingly, lactate itself upregulated the expression of GPR81 in MDA-MB-231 cells (Fig. 1c). These results show that GPR81 expression is increased in breast cancer cells and, thus, suggest that GPR81 plays a role in the regulation of lactate metabolism and is associated with aggressiveness of breast cancer.
Knockdown of GPR81 suppressed lactate secretion and tumor growth. To further determine the role of GPR81 in breast cancer, GPR81 was stably silenced using short hairpin (sh)RNA in MDA-MB-231 cells, which showed the highest expression of GPR81 ( Fig. 1a) (hereafter designated shGPR81 cells). We established two MDA-MB-231 lines in which GPR81 was stably suppressed: shGPR81 #1 and shGPR81 #2. We confirmed by RT-qPCR and western blotting that the expression of GPR81 was significantly reduced in shGPR81 #1 and shGPR81 #2 cells compared with that in cells expressing the control hairpin RNA (shNT) (Fig. 2a,b and Supplementary Fig. S1). Of interest, knockdown of GPR81 also decreased the expression of MCT4 (Fig. 2a,b and Supplementary Fig. S1). Furthermore, shGPR81 #1 cells showed decreased lactate release into the culture media compared with that of shNT cells (Fig. 2c).
Next, we examined the role of GPR81 in cancer cell proliferation. Proliferation of shGPR81 cells was significantly reduced compared with that of shNT cells as assessed by the WST-1 assay ( Fig. 3a and Supplementary  Fig. S1). Furthermore, anchorage-independent growth of shGPR81 cells was also reduced (Fig. 3b). Importantly, when the shNT or shGPR81 cells were implanted subcutaneously in nude mice, the tumor growth of the shGPR81 cells was substantially decreased compared with that of the shNT cells ( Fig. 3c and Supplementary Fig. S1).
We then examined whether glycolytic ATP production was affected by silencing of GPR81 expression. Lactate treatment increased the ATP production in shNT cells, which was attenuated in shGPR81 cells, suggesting that GPR81 regulated lactate-dependent ATP production (Fig. 4a). Mechanistically, shGPR81 cells showed decreased expression of rate-limiting glycolytic enzymes, including hexokinase 2 (HK2), PFK1, and LDHA, compared with those in shNT cells (Fig. 4b). These data collectively suggest that GPR81 regulates glycolytic ATP production and tumor growth in breast cancer cells. However, it should be noted that reduced expression of the lactate transporters MCT4 and MCT1 may also contribute to decreased ATP production in shGPR81 cells. Breast  www.nature.com/scientificreports/ metastasis 28 . Thus, we next determined whether GPR81 plays a role in the regulation of tumor growth in bone. We injected shNT or shGPR81 cells in the hind limbs of female nude mice and evaluated tumor growth by quantifying the osteolytic lesions by X-ray analysis 29 . We found that the development of osteolytic lesions by shGPR81 cells was significantly decreased compared with that of shNT cells (Fig. 5a,b and Supplementary Fig. S2). Histological analysis further demonstrated that GPR81 was strongly expressed in shNT tumors ( Supplementary  Fig. S3), and the number of tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated osteoclasts at the tumor-bone interface was significantly decreased in shGPR81 cell-injected bone compared with that of shNT cell-injected bone (Fig. 5c-e). Moreover, we found that knockdown of GPR81 decreased the expression of interleukin-6 (IL6) and IL11 mRNA, whereas no significant change was observed for parathyroid hormone-related protein (PTHrP) mRNA expression, contrary to our expectation (Fig. 5f). These data suggest that GPR81 plays an important role in the development of osteolysis and breast tumor growth in bone.

GPR81 controls cancer cell motility.
To further investigate the role of GPR81 in cancer aggressiveness, we performed comparative global gene expression analysis after RNA-sequencing (RNA-seq) of shNT and shGPR81 cells. RNA-seq analysis identified 261 downregulated and 152 upregulated genes using a threshold for false discovery rate (FDR) of < 0.05 and a fold-change threshold > 1.5 (Fig. 6a). Next, we performed Gene Ontology (GO) enrichment analysis for molecular function and found that shGPR81 cells were significantly enriched for downregulated genes from the positive regulation of cell motility gene set (GO:2000147) (Fig. 6b, Supplementary Table S1). Several genes associated with cancer cell motility, including SEMA5A, PDGFRB, TGFB2, CXCR4, SNAI1, ICAM1, and NEDD9, were decreased in shGPR81 cells (Fig. 6c).
To investigate whether GPR81 regulated cancer cell migration and invasion, we performed wound-healing and cell invasion assays. As expected, the migration and cell invasion activities of the shGPR81 cells were impaired compared with those of shNT cells (Fig. 7a,b and Supplementary Fig. S4). These data suggest that GPR81 contributes to cancer cell invasion and migration. GPR81 is highly expressed in clinical samples of human breast cancer. Finally, we determined the clinical relevance of GPR81 expression in human breast cancers using tissue microarrays. Immunohistochemi-  Table 1). GPR81 was not detected in adjacent normal breast tissue, while white adipocytes expressed GPR81 (Fig. 8b). In contrast, stromal cells near the breast cancer tissue showed weak expression of GPR81 ( Supplementary Fig. S5). These results suggest that the expression of GPR81 is associated with the degree of malignancy of human breast carcinoma.

Discussion
It is well established that cancer cells produce abundant lactate through aerobic glycolysis to fuel sustained tumor growth and secrete lactate into the tumor microenvironment 7 . Despite being exposed to high concentrations of lactate, it remains unclear whether cancer cells sense extracellular lactate and subsequently control their own behavior and energy metabolism. In this study, we demonstrated that activation of GPR81 in breast cancer cells by lactate impacted diverse pathological processes, including aerobic glycolysis, cell proliferation, and cell motility. In addition, histological examination showed rich GPR81 expression in clinical samples of breast tumors. Our findings suggest that GPR81 regulates breast cancer aggressiveness and, thus, is a potential therapeutic target for breast cancer. We demonstrated that silencing GPR81 inhibited cell proliferation and tumor growth. It is likely that GPR81 is important for cell proliferation by regulating multiple pathways of energy metabolism. Several key enzymes of glycolysis were decreased after silencing GPR81; therefore, GPR81 is involved in the regulation of the glycolytic pathway. Our data also showed that the expression of the lactate transporter MCT4 was decreased in shGPR81 cells. Cancer cells are reported to release and absorb lactate through MCTs and utilize lactate as an energy source under glucose-deficient conditions 16 . GPR81 likely controls lactate transport and subsequent ATP production. Interestingly, recent evidence has shown a critical role for MCTs in tumor development, and MCT1 inhibitors have undergone clinical trials as potential anti-cancer treatments 30,31 . Moreover, Brown et al. reported that tumor-derived lactate activated GPR81 in dendritic cells and, consequently, promoted immune cell evasion by inhibiting tumor-specific antigen presentation 26 . These data collectively suggest that GPR81 signaling in cancer and immune cells is pivotal for tumor growth, and GPR81 may be a promising anti-cancer target. Although the development of antagonists for GPR81 is in the developmental phase thus far, it will be valuable to design and test pharmacological inhibitors of GPR81.
Our results suggest that inhibition of the lactate-GPR81 axis impairs cell motility. It is intriguing that several studies have reported that aerobic glycolysis regulates cell motility 32  www.nature.com/scientificreports/ glycolysis is the primary energy source for cancer cell motility, and motility was attenuated by the inhibition of glycolysis but not by the inhibition of mitochondrial ATP production 32 . Moreover, hypoxic conditions promoted cell migratory activity by regulating RhoA-ROCK1 expression 33 . These data demonstrate the interplay between lactate, glycolysis, and cancer cell motility, and our findings suggest GPR81 may be an essential molecule that mediates this interplay 33 . In addition to cell motility, knockdown of GPR81 significantly decreased epithelial-mesenchymal transition-associated genes, including SNAI1, NEDD9, and TGFB2. These data collectively indicate that GPR81 regulates metastatic activity of cancer. Further studies are warranted to determine the role of GPR81 in cancer metastasis. Breast cancer cells frequently metastasize to bone, and MDA-MB-231 cells have been used to investigate osteolytic bone metastasis of breast cancer in vivo 34 . Our data suggested that the knockdown of GPR81 impaired osteolysis and tumor growth partly because of a decrease in the expression of osteolytic cytokines, including IL-6 and IL-11. Both clinical and preclinical studies have reported that IL-6 and IL-11 released from breast cancer cells contributed to the development of bone metastasis [35][36][37] . Although the molecular mechanism by which GPR81 regulates IL-6 and IL-11 expression remains unclear, it is plausible that lactate controls the production of these cytokines through GPR81 in breast cancer cells. In support of this concept, Gene Ontology analysis of RNA-seq data demonstrated that genes associated with cytokine production (GO:0001819: Positive regulation of cytokine production) were significantly downregulated in shGPR81 cells. Because cytokines are known to regulate diverse processes during tumor progression, it would be of interest to determine the role of GPR81 in cytokine-dependent cancer malignancy.   38 . Osteoclasts incorporate lactate that is released from breast cancer cells through MCT1 and use this lactate as a fuel to increase oxidative metabolism, thereby promoting bone resorption 39 . Because our results showed that GPR81 regulated MCT4 expression, these data suggest that GPR81 may indirectly control osteoclast function. Interestingly, it has been reported that lactate controls the inflammatory processes associated with macrophages, which have the potential to differentiate into osteoclasts [40][41][42] . Expression and function of GPR81 in osteoclasts need to be extensively studied in the future.
Previous studies have established that activation of GPR81 by lactate represses cAMP production through G(i) protein-dependent inhibition of adenylyl cyclase activity 20,[22][23][24] . Importantly, the accumulation of intracellular cAMP has reportedly shown anti-tumor activity in some types of cancer cells. For instance, natural cAMPelevating compounds, such as forskolin, have been reported to inhibit cell proliferation and migration and induce apoptosis in cancer cells [43][44][45] . Given that lactate-dependent activation of GPR81 suppressed intracellular cAMP levels, we propose that silencing GPR81 increased the accumulation of cAMP, which resulted in the various types of anti-tumor effects observed in MDA-MB-231 cells. However, it should be noted that cAMP signaling has been reported to both promote and suppress tumor function 46 . Determining correlations between GPR81dependent intracellular signaling and anti-tumor effects will be an important subject for further investigation.
Although GPR81 is strongly expressed in human breast cancer, the underlying mechanism that controls GPR81 expression in breast cancer remains unclear. Recently, Xie et al. reported that lactate itself promoted GPR81 expression by activating the STAT3 pathway in lung cancer 47 . Lactate also increased GPR81 expression in dendritic cells and promoted immunosuppression 26 . Thus, it would be reasonable to speculate that cancer cells produce and sense lactate through GPR81 to reprogram energy metabolism. However, because lactate mainly accumulates in hypoxic areas, and lactate concentration is not uniform within tumors, other characteristic features of cancer, including low pH, hypoxia, and low nutrients, are likely involved in GPR81 expression. The different expression levels of GPR81 contribute to the heterogeneity of cancer cells. Further analyses to uncover the mechanisms involved in GPR81 expression will be necessary to better understand the role of GPR81 in cancer.
Our results showed that knockdown of GPR81 decreased cancer cell aggressiveness. However, GPR81 knockdown decreased MCT1 and MCT4 expression, and impaired cancer cell aggressiveness observed in shGPR81 cells may have resulted from reduced lactate usage because of decreased MCT expression. Furthermore, pharmacological inhibitors against GPR81 were not tested in this study. A specific GPR81 antagonist that does not affect MCT1 and MCT4 expression will be a useful tool to address mechanistic questions. However, GPR81 antagonists are unavailable at present. Several studies have used 3-hydroxybutyric acid (3-OBA) as an antagonist against GPR81, and 3-OBA treatment showed approximately equivalent effects compared with that of GPR81 knockdown 48,49 ; however, there is no experimental evidence that shows specific antagonistic effects of 3-OBA on GPR81 50 . Development of a GPR81-specific antagonist and determining its role in cancer cell activity await further investigation.
In conclusion, our study demonstrates that GPR81 controls tumor activity by regulating lactate transport and glycolytic metabolism in breast cancer cells. We propose that GPR81 may be a novel therapeutic target for breast cancer. Western blotting. The cell lysates were mixed with 4 × Laemmli sample buffer (Bio-Rad) and heated at 95 °C for 5 min. The proteins were separated using SDS-PAGE (7.5%-10% gels) and transferred to nitrocellulose membranes (GE Healthcare). After blocking, the membranes were incubated with primary antibodies overnight at 4 °C and HRP-conjugated secondary antibodies for 1 h. Proteins were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgGs using an enhanced chemiluminescence detection kit (Immunostar LD; WAKO; Osaka, Japan). Anti-GPR81 (#NLS2095, 1:1000) was purchased from Novus Biologicals (Littleton, CO, USA).  RT-qPCR. Total RNA from cells was extracted using the NucleoSpin RNA Plus total RNA isolation system (Macherey-Nagel GmbH & Co.; Düren, Germany). First-strand cDNAs were synthesized using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Ltd., Osaka, Japan). Quantitative real-time reverse transcription-PCR analysis was performed using the Standard Taq PCR protocol, SYBR Green PCR protocol, and a 7300 Real-  www.nature.com/scientificreports/ Proliferation assay. The cell proliferation assay was performed using the Premix WST-1 Cell Proliferation Assay System (Roche Holding AG, Basel, Switzerland) in accordance with the manufacturer's protocol. Briefly, MDA-MB-231 cells containing shNT or shGPR81 RNA were plated in 24-well plates (40,000 cells/well) and incubated at 37 °C in a 5% CO 2 atmosphere. On days 1 and 2, cell proliferation reagent was added to each well and incubated for 1 h. The absorbance was measured using the Model 550 micro-plate reader described above.
Animal experiments. All  TRAP staining. Bone sections were stained for TRAP using the Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma-Aldrich) and analyzed for osteoclastic bone destruction using a light microscope (Leica Microsytems). TRAP + osteoclasts were counted at the tumor-bone interface of the endocortical bone using three fields of view per section for each sample as previously reported 53  RNA-seq data were analyzed using iDEP (integrated Differential Expression and Pathway analysis) 54 . Briefly, read count data from three replicates each of shNT and shGPR81 expressing cells were generated and uploaded to the iDEP website (http:// bioin forma tics. sdsta te. edu/ idep/). Differentially expressed genes were identified using the following thresholds: FDR < 0.05 and minimal fold change > 1.5. Gene Ontology enrichment analysis for molecular function was performed using the Metascape 55 website (https:// metas cape. org/ gp/ index. html#/ main/ step1). The raw data have been deposited in the NCBI Gene Expression Omnibus database (GSE186211).
Wound-healing assay. For the wound-healing assay, shNT and shGPR81 cells were cultured for 24 h in DMEM containing 10% FBS in 6 well plates. After confirming that a complete monolayer had formed, the monolayers were wounded by scratching a line through the cellular layer with a standard 200-μL plastic pipette tip. Migration was observed throughout the wound area after 24 h using a phase-contrast microscope equipped with a camera, and migration distances were measured using the photographic images as previously described 56 . Migration assay. Cell invasion assays were performed using a Cell Invasion Assay kit (Cell Biolabs Inc.; San Diego, CA, USA) in accordance with the manufacturer's instructions. Briefly, MDA-MB-231 cells expressing shNT or shGPR81 RNA were suspended in serum-free media. The cells (1.5 × 10 5 /chamber) were placed in inserts designed for a 24-well plate. Each insert contained a thin layer of extracellular matrix over a polycarbonate membrane (8 µm pore size). Lower compartments were filled with DMEM containing 10% FBS. After incubation for 6 h at 37 °C in a 5% CO 2 incubator, cells that invaded and migrated through the matrix-containing membrane and reached the lower surface of the invasion chamber were observed using a phase-contrast microscope with an attached camera. The cells that had migrated were counted using photographic images.
Tissue microarray. GPR81 expression in invasive human breast carcinoma and normal breast tissues was determined using tissue microarrays (BR245b, BR246b, and BR246d; US Biomax, Inc.; Rockville, MD, USA). The antigen was activated by heating in a citric acid solution. For immunohistochemical analysis of tissues, specimens were incubated with anti-GPR81 antibody (1:100, Novus) overnight at 4 °C, followed by treatment with streptavidin-biotin complex (1:100, EnVision + System-HRP Labelled Polymer; Dako Cytomation; Carpinteria, CA, USA) for 60 min. The tissues were visualized using the Liquid DAB + Substrate Chromogen System (Dako). Statistical analysis. Randomization and blinding were not performed in the animal studies. Data were statistically analyzed by Student's t-test for comparisons between two groups. For more than two groups, we used www.nature.com/scientificreports/ one-way analysis of variance (ANOVA) or two-way ANOVA followed by Tukey's post hoc test. P-values < 0.05 were considered statistically significant.
Ethics. Animal experimentation was performed in strict accordance with the guidelines for proper conduct of animal experiments and related activities of Osaka University Graduate School of Dentistry. All animals were handled in accordance with the protocol approved by the Animal Committee of Osaka University Graduate School of Dentistry.

Data availability
RNA-seq data have been deposited in the NCBI Gene Expression Omnibus (https:// www. ncbi. nlm. nih. gov/ geo/) under accession number GSE186211.